A Sensitive Green-Red FRET Sensor for Calpain-1 Utilizing Heterodimeric Fluorescent Proteins from Distinct Evolutionary Origins

  • ACS Sens. 2026 Mar 27;11(3):1891-1907. doi: 10.1021/acssensors.5c02811.
Fangfang Yang  1 ,  Mengying Deng  1 ,  Xiaorong Pan  2 ,  Jintao Wang  3 ,  Pei Huang  4 ,  Zirui Ou  5 ,  Xiaoyu Zou  5 ,  Fahim Muhammad  3 ,  Shiyi Gan  6 ,  Qixing Yang  3 ,  Cuihan Shi  3 ,  Jingya Chen  1 ,  Junfeng Li  7 ,  Yongjuan Zhao  8 ,  Pei-Gen Ren  7 ,  Xinan Liu  9 ,  Yang Du  6 ,  Tong Wang  2 ,  Zuxin Chen  3 ,  Jun Chu  1  10  11
Affiliations
  • 1. Research Center for Primate Neuromodulation and Neuroimaging, Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences (CAS), Shenzhen 518055, China.
  • 2. The Brain Center, School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China.
  • 3. Shenzhen Key Laboratory of Drug Addiction, Shenzhen Neher Neural Plasticity Laboratory, the Brain Cognition and Brain Disease Institute, SIAT-CAS; Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen 518055, China.
  • 4. School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China.
  • 5. College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China.
  • 6. Kobilka Institute of Innovative Drug Discovery, Shenzhen Key Laboratory of Steroid Drug Discovery and Development, School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, China.
  • 7. Institute of Biomedicine and Biotechnology, SIAT-CAS, Shenzhen 518055, China.
  • 8. School of Medicine, The Chinese University of Hong Kong Shenzhen, Shenzhen 518172, China.
  • 9. Guangdong Provincial Key Laboratory of Brain Connectome and Behavior, CAS Key Laboratory of Brain Connectome and Manipulation, Brain Cognition and Brain Disease Institute (BCBDI), SIAT-CAS, Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen 518055, China.
  • 10. Shenzhen Key Laboratory for Molecular Imaging, Guangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology, SIAT-CAS, Shenzhen 518055, China.
  • 11. Key Laboratory of Biomedical Imaging, SIAT-CAS, Shenzhen 518055, China.
PMID: 41837894 DOI: 10.1021/acssensors.5c02811
Abstract

Calpain-1, a calcium-dependent cysteine protease, plays a pivotal role in various biological functions. However, real-time monitoring of its activity has been limited by the lack of high-performance calpain sensors. Here, we report the development of iFoCAL, a sensitive and specific fluorescence resonance energy transfer (FRET) sensor for calpain-1, which employs mClover3 and mScarletX, a newly engineered green-red FRET pair with weak interactions. Using iFoCAL, we revealed distinct spatiotemporal patterns of calpain-1 activation: gradual and global activation in whole cells during endoplasmic reticulum (ER) calcium release, fast and focal activation in axons during mechanical stress, and region-specific activation in a Parkinson's disease model. These findings provide the first direct evidence of distinct calpain-1 dynamics across diverse cellular contexts.

Keywords
FRET; Parkinson’s disease; axonal injury; calpain-1; fluorescent protein.
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